Lecture 3 (January 14) - Copy (1) PDF

Terminology from Lecture 2 1. Derisking a projects: Examples: genetic evidence (for target) or biomarker (on-target) 2. Reproducibility as a factor in project selection (Novel (published science)-First step: reproduce result 3. Assays: Biochemical (for example isolated enzyme), in vitro (cellu...

Terminology from Lecture 2 1. Derisking a projects: Examples: genetic evidence (for target) or biomarker (on-target) 2. Reproducibility as a factor in project selection (Novel (published science)-First step: reproduce result 3. Assays: Biochemical (for example isolated enzyme), in vitro (cellular) and in vivo (animal studies) 4. Central Dogma of Biology 5. Endogenous small molecules: steroids, eicosanoids (such as prostaglandins and thromboxane) 6. GWAS Genome-Wide Associated Studies, identify SNPs (single nucleotide polymorphisms (disease associaiton 7. PheWAS Phenotype Wide Associated Studies - common disease phenotype-identify common SNPs? 8. ADME/PK Absorption Distribution, Metabolism and Excretion. Pharmacokinetics 9. Urinary/Renal System Important in lead optimization (drug distribution, clearance and metabolism) 10. Gene therapy, CRISPR/Cas9 first marketed drug Casgevy for treatment of Sickle Cell Disease 11. RNAi (interfering RNA) Use in target validation and therapeutic strategy 12. Notable Nobel prizes in chemistry and medicine-physiology 13. Chemical Genetics Small molecule (endogenous or SM drug) modulate macromolecular (Protein, DNA, RNA) 14. Reverse Chemical Genetics (target screen-protein-HYPOTHESIS based) and Forward Chemical Genetics (phenotype screen, hypothesis seeking, and possibly target Identifying; cf. Aspirin, Morphine, Penicillin) Compare to classical genetics random (error prone PCR or chemical) versus targeted mutagenesis (targeted knockout or CRISPR) 15. Chemical space-its big (ca. 1060 for SM with MW below 500) Recommended Reading: p. 38-43 (CC&K) 1 2  Target Hit Lead Lead Candidate IND Identification Identification Identification Optimization Profiling Enabling I. Target Identification/Selection n Disease Association: Basic science and clinical knowledge (GWAS or PheWAS) n Most human diseases (aside from infection or injury) involve disruption of homeostasis (that is steady state of biomolecular elements-proteins, metabolites, glucose, reactive oxygen species, etc) n Loss of homeostatsis is often associated with gain or loss of “function” of signaling pathways and/or associated proteins such as enzymes n Small molecule drugs serve to increase or decrease function with the aim of returning to homeostatsis n Mechanism of Action (MOA): “Functional” pharmacology, biomarker (cellular and in vivo) II. Target Validation n Is the target “druggable”? Can it be modulated by a small molecule? On-target? (for example…Biomarker and Chemical Biology) n Complete validation is a marketed drug 3 Human Genome Project (2023)-See Ref. 1, Chapter 3 (BEB) n 23 Chromosomes; over 3 billion DNA base pairs n Encoding ca. 20,000 to 25,000 Proteins (not all involved in disease progression) n Estimated 5000 potentially ”druggable” macromolecular targets n Plus ca. 3200 possibly suitable targets for biological therapeutics n Consider possible targets for infectious diseases: microbial genomes are much smaller Classical (macromolecular) Targets for Disease Intervention n Enzymes For example, cyclooxygenase (COX), Aspirin n G-protein-coupled receptors (GPCRs) Cell Signaling (34% FDA Approved SM Drugs act on GPCRs). For example, Zantac for treating acid reflux n Ion channels For example, Lidocaine blocks sodium ion channels n Transporters Antidepressants such as Prozac and Celexa target serotonin transporter (SERT) resulting increase of serotonin by blocking its uptake Drug Repurposing and Drug Repositioning n An alternative to full Drug Discovery and Development n From DrugBank 5.1.2 (December 2018): Over 2,000 FDA approved drugs and >11,000 through Phase 1 clinical trials (passed Safety and Tolerability n From BEB (Figure 3.2) 1204 small molecule drugs and 166 biologics 4 Drug Repurposing Drug repurposing is a strategy for identifying new uses for approved drugs that are outside the scope of the original medical indication. Drug Repositioning Drug repositioning is a strategy for identifying new uses for investigational drugs (IND) that are outside the scope of the original medical indication. 5 5.Drug Repurposing FDA Approved & Marketed 3. Drug Repositioning IND Filed and Approved for Clinical Investigation 6 Drug Repurposing & Repostioning at VUMC-BioVU Supported by Human Genetics How we got from 10,505 drugs to 237… Biologics removed Toxics (such as chemotherapy agents like daunorubicin were removed) Non-mammalian targets-antibiotics…penicillin, antiviral, antifungal removed Known Mechanism of Action; only one target (for example, some kinase inhibitors hit more than one kinase…) SM-Target (primarily protein) Only one SM of a particular target represented (such as ACE inhibitors) Challa AP, Lavieri RR, et al. Systematically Prioritizing Candidates in Genome-Based Drug Repurposing. Assay Drug Dev Technol. 2019 Nov/Dec;17(8):352-363. 7  Clinical and Patient Data in BioVU-Genotyping We have clinical data available before: The first dose of an investigational new drug is ever given A new preclinical program is launched Knowledge of “healthy” individuals equally valuable 8 8  Problem Statement (PheWAS) time = 6-12 months No radiographic Metastatic recurrence evidence of residual disease 9  PheWAS Shows Metastatic Recurrence is associated with SNP in Thromboxane A2 Receptor (TBA2) – G-Protein Coupled Receptor (GPCR) Threonine 399 to Alanine has been associated with metastatic recurrence in cancer patients This is a ”gain of function” disease Need drug (SM) to reduce function by TBA2 receptor Image: Huang, J.S. et al. Cellular Signaling, Volume 16, Issue 5, May 2004 10  T399A interferes with receptor desensitization Image: Huang, J.S. et al. Cellular Signalling, Volume 16, Issue 5, May 2004 11  Thromboxane A2 receptor is a G-protein coupled receptor (GPCR) – a membrane associated protein GPCRs are often regulated by an endogenous small molecule (including peptides etc)-how and what regulates some GPCRs is not known- these are sometimes referred to as ”orphan GPCRs” The site of small molecule binding leading to regulation is called the orthosteric site (in contrast an allosteric site differ from the site of “action/regulation” Agonists are small molecules that bind to ”a receptor” on a cell and “activate it” triggering a biological response Antagonists are small molecules that bind to “a receptor” and block a biological response Thromboxane A2 (TXA2) is the endogenous ligand of the thromboxane A2 receptor – TXA2 in this role is an agonist (turns on signaling) Thromboxane A2 has a half-life of 30 seconds! Rapidly converting TXA2 to TXB2 Function TXA2 receptor: Hemostasis: Interacts with thromboxane A2 (TXA2) to cause platelets to clump together Thromboxane A2 has been linked to diseases like myocardial infarction, stroke, and bronchial asthma ANTAGONISTS of TXA2 receptor have been developed for treatment of these associated diseases One drug that entered the clinic: Ifetroban COOH COOH COOH OH t1/2 30 sec O O H 2O O HO O N O N OH OH H O thromboxane A2 thromboxane B2 ifetroban 12 Drug: ifetroban design based upon endogenous TXA2 Thromboxane A2 is a receptor angonist Ifetroban is a receptor antagonist Developed at Bristol Myers Squibb Note: similarities in structure: carboxylic acid, “cis olefin”, bicyclic oxygenated core and hydrogen bond donor (OH and NH) Going from an agonist to antagonist by structural modification Clinical studies for cardiovascular disease Through phase I (active phase II for other therapeutic areas) Safe and well tolerated COOH COOH O O N O O N OH H O thromboxane A2 ifetroban AGONIST ANTAGONIST endogenous ligand medicinal chemistry derived 13  Thromboxane A2 Receptor Signaling 3D structure of the Thromboxane Prostanoid Rececptor (TPr) bound to various TPr antagonists Fan, H, et al. Structural basis for ligand recognition of the human thromboxane A2 receptor. Nat Chem Biol, 2019. 14  Ifetroban decreases metastasis in a spontaneous mouse model of TNBC metastasis Ifetroban also significantly reduces metastasis in additional cancer models and cancer types: üSpontaneous mouse models of TNBC metastasis: Neoadjuvant treatment Adjuvant treatment üMouse models of experimental metastasis using: Mouse and human breast cancer cell lines Human pancreatic and lung cancer cell lines Werfel TA, et al. Repurposing of a Thromboxane Receptor Inhibitor Based on a Novel Role in Metastasis Identified by Phenome-Wide Association Study. Mol Cancer Ther. 2020 Dec;19(12):2454-2464. 15  Ifetroban blocks trans-endothelial migration of tumors cells Werfel TA, et al. Repurposing of a Thromboxane Receptor Inhibitor Based on a Novel Role in Metastasis Identified by Phenome-Wide Association Study. Mol Cancer Ther. 2020 Dec;19(12):2454-2464. 16  4T1 mouse model data support the predicted impact of Ifetroban on metastasis Decreasing the ability of tumor cells to detach from the vascular epithelium and attach to platelets Increasing exposure of circulating tumor cells to host immune Preventing colonization by affecting P-selectin-mediated interactions 17 responses  commentary Target validation using chemical probes Mark E Bunnage, Eugene L Piatnitski Chekler & Lyn H Jones Fully profiled chemical probes are essential to support the unbiased interpretation of biological experiments necessary for rigorous preclinical target validation. We believe that by developing a ‘chemical probe tool kit’, and a framework for its use, chemical biology can have a more central role in identifying targets of potential relevance to disease, avoiding many of the biases that complicate target validation as practiced currently. M Quality © 2013 Nature America, Inc. All rights reserved. edicinal chemistry chemical design and synthesis can provide selective tool probes whereas the successfulfor programs unbiased achieved what is termed the ‘three pillars of survival’: action;interpretation of biological experiments pillar 2, proof of target engagement; and pillar 3, expression of functional compounds to interrogate biology, pillar 1, sufficient exposure at the site of pharmacological activity. Moreover, for the 3 thus Rigorous illustrating preclinical the synergy between chemical biology and drug discovery. A 1,2 target validation Exposure at Target Functional Relevant site of action engagement pharmacology phenotype major augment issue with using small molecules to Chemical target validation beforebiology launching enabled identification of targets of potential relevance to a full drug discovery program is having the disease confidence that we have effectively validated the target of interest in a relevant phenotypic assay. There are many widely used chemical probes Many that do not meet examples generally accepted potency and selection criteria, and the of chemical probes that do NOT meet potency and selection 1 2 3 4 criteria conclusions made from their use are suspect. Similarly, the use of selective chemical probes in heavily manipulated biological assays Heavily manipulated is less likely to generate of relevance to human disease. information biological assays NOT likely to be relevant to human disease When considering this problem, we npg can directly draw from our experiences in For example, use of LC-MS to measure For example, activity- based proteomic Proximal biomarker, such as phosphorylated Specific effects, such as translocation, clinical drug development. A retrospective intracellular profiling, fluorescent products of kinases, development, analysisAnalysis of 44 drug programs in phase II trials at Pfizer showed failure resulted of 44 drug programs in phase 2 clinical trials at Pfizer revealed that most concentrations or radiolabeled probes other PTMs morphology, size failures resulted from a lack of efficacy, Figure 1 | The four pillars of cell-based target validation using chemical probes. from a lack of efficacy. Successful programs achieved the three pillars of survival; Box 11. | The Exposure at site of action; 2. Proof of target engagement; 3. functional four pillars of target validation. pharmacology; 4. Relevant phenotype (disease related) Pillar 1. Exposure at the site of action. Experiments should confirm that the probe is able to achieve pharmacologically relevant concentrations inside unbiased selectivity determination in a more physiologically relevant environment. or at the cell. ‘Mismatches’ between the biochemical activity of a probe (for example, inhibition of recombinant enzyme activity) and whole-cell Pillar 3. Expression of functional pharmacology. Assays can be created that activity are often explained by invoking poor cell permeability, but usually measure the pharmacology of the probe, often assessing a proximal biomarker there are no data to confirm this hypothesis. Analytical techniques can be for activity, for example, the phosphorylated product of a kinase, an acetylated used to measure intracellular (and possibly subcellular) concentrations of histone tail or depletion of an HSP90 client protein. a probe. Bunnage, Chekler and Jones (Pfizer) Nature Chem. Bio. 2013, 195 18 Pillar 4. Proof of phenotype perturbation. The challenge for cell biology is to Pillar 2. Target engagement. This is the most technically challenging pillar, create assays that capture the most relevant phenotypic changes in the though it is essential to link exposure at the site of action (pillar 1) to context of human disease and for which there is a high degree of confidence  can directly draw from our experiences in LC-MS to measure based proteomic such as phosphorylated such as translocation, clinical drug development. A retrospective intracellular profiling, fluorescent products of kinases, development, concentrations or radiolabeled probes other PTMs morphology, size analysis of 44 drug programs in phase 2 clinical trials at Pfizer revealed that most The Four Pillars of Cell-Based Target Validation failures resulted from a lack of efficacy, Figure 1 | The four pillars of cell-based target validation using chemical probes. Box 1 | The four pillars of target validation. Pillar 1. Exposure at the site of action. Experiments should confirm that the unbiased selectivity determination in a more physiologically relevant probe is able to achieve pharmacologically relevant concentrations inside environment. or at the cell. ‘Mismatches’ between the biochemical activity of a probe (for example, inhibition of recombinant enzyme activity) and whole-cell Pillar 3. Expression of functional pharmacology. Assays can be created that activity are often explained by invoking poor cell permeability, but usually measure the pharmacology of the probe, often assessing a proximal biomarker there are no data to confirm this hypothesis. Analytical techniques can be for activity, for example, the phosphorylated product of a kinase, an acetylated used to measure intracellular (and possibly subcellular) concentrations of histone tail or depletion of an HSP90 client protein. a probe. Pillar 4. Proof of phenotype perturbation. The challenge for cell biology is to Pillar 2. Target engagement. This is the most technically challenging pillar, create assays that capture the most relevant phenotypic changes in the though it is essential to link exposure at the site of action (pillar 1) to context of human disease and for which there is a high degree of confidence pharmacology (pillar 3) and phenotype (pillar 4). Exciting advances in in their ‘translatability’. Phenomena that may lead to false positive results, techniques such as activity-based proteomics have enabled the such as nonspecific cell death, should be ruled out at an early stage. The quantification of target engagement to be made. Sophisticated functional strongest rationale for target validation can be provided if all four pillars are probes can measure occupancy inside the cell and facilitate built in the same pathophysiologically relevant cell system. NATURE CHEMICAL BIOLOGY | VOL 9 | APRIL 2013 | www.nature.com/naturechemicalbiology 195 Pillar 1: Exposure at the site of action: Biochemical versus in cell activity. Possible to detect small molecule (probe) in cell and/or cell organelle by Mass Spec (cell permeability and efflux) Pillar 2: Target Engagement. In cell thermal shift assay (protein target mobility depends on small molecule (probe) engagement). Activity Based Protein Profiling (ABPP) Pillar 3: Functional pharmacology Biomarker – in this example, protein phosphorylation, protein concentration Pillar 4: Proof of phenotype perturbation Translatability-”disease relevance” The Four Pillars of Cell-Based Target Validation  Quality Probe for Target ”Validation: Selectivity, potency, control compounds, multiple scaffolds, Physiochemical and commentary DMPK properties Another Central relatedto probe aspect of pillar 1 is Selectivity ensuring that exposures are commensurate >100-fold withdevelopment the on-target activity ofis the probe and not in great excess, such that selectivity collaboration windows over off-targets areamong eroded. This is important as the use of probes in cell Inactive control (for example, Multiple active and selective pharmacologists, biology experiments at concentrations biologists, in enantiomer) chemical classes and chemists (analytical, excess of their selectivity window could lead to erroneous links being made between synthesis, on-target medicinal activity and phenotypes, when and may be the off-target activity that is actually it Potency Structure